The potential of genomics to identify genes important for alcoholism has not been completely realized because ethanol produces a surfeit of changes in gene expression, most of which do not seem to make any meaningful contribution to the ethanol response of interest. A simple and effective way to pan for genomic changes relevant for an alcoholism phenotype would be invaluable. The problem is solved by focusing on genes that show common changes produced by two chemically and metabolically dissimilar drugs, ethanol and benzyl alcohol, which produce cross-tolerance by a mechanism with a shared molecular origin. Genes that respond in the same way to both alcohols will be identified by genomically surveying epigenetic histone modifications and gene expression. These will be functionally tested by mutant analysis in the fruit fly Drosophila melanogaster, an animal that has ethanol responses remarkably similar to humans. The alcohol response genes will be tested for a functional role in ethanol tolerance and two withdrawal/dependence symptoms - alcohol withdrawal induced hyperexcitability and cognitive ethanol dependence. Parts of this response have already been shown to be conserved in mammals. To directly bridge our work to human alcoholism the data will be collaboratively compared to human alcoholic datasets. The advantage of using Drosophila is that candidate genes can be rapidly and functionally tested by mutant analysis for their role in producing these ethanol responses. In Aim 1, chromatin and RNA will be prepared from the heads of flies treated with ethanol or benzyl alcohol. Epigenetic and gene expression changes common to both drugs will be identified by ChIP-seq to identify changes in histone H3 and H4 acetylation and methylation (H3K36me1 and H3K36me3). These histone marks have tight linkage to changes in gene activity. RNA-seq will be used to monitor changes in gene expression. These candidates and pre-existing candidates from an epigenetic screen will be tested in Aim 2 and 3 by mutant, RNAi, and mis-expression analysis. In Aim 2, candidate genes will be tested for a role in functional tolerance to ethanol sedation. In Aim 3, th effects of mutations on alcohol-withdrawal will be determined electrophysiologically in adults and behaviorally in larvae. Adult flies show alcohol-withdrawal hyperexcitability that increases the susceptibility for seizures. While in ethanol-adapted larvae, the capacity for associative learning declines during withdrawal and is restored after ethanol reinstatement-demonstrating functional dependence. This contribution will be significant because it will define the origins of functional tolerance phenotype and establish its relationship to dependence phenotypes. The proposed research is innovative because it uses 1) a shared drug response to focus a genomic survey on ethanol-response genes, 2) epigenetic histone modifications to help identify genes that transcriptionally respond to ethanol intoxication, and 3) new behavioral assays that allow us to detect tolerance and physiological dependence in flies. Over its 5 year span, this project will be used to train 2 post-docs, 2 graduate (Ph.D) and 10 undergraduate students.